The long term goal of this proposal is to understand the molecular machinery involved in intracellular membrane transport and secretion in neural, endocrine, and exocrine cells. Most cells have the capacity to secrete proteins, but not all cells utilize the same machinery. In particular, secretion from mammalian cells can take many different forms. The different pathways enable the cells to externalize molecules in a timely and spatially regulated fashion. These pathways together control a variety of cellular processes, including plasma membrane growth, cell-cell communication, and regulation of cell surface transport activities. Currently, little is known about the molecular components controlling export from each pathway, and how the machinery differ from one another. Neuroendocrine cells will be used as a model system to study two secretory pathways: a constitutive pathway and a regulated pathway. Exocytotic vesicles involved in these two secretory pathways are formed from the same organelle, the trans-Golgi network. Thus, our studies will be aimed at understanding the mechanisms for transport between the trans-Golgi network and the cell surface along both paths. The specific goals of these studies are: (1) to elucidate the mechanism for the formation of constitutive and regulated vesicles from the trans-Golgi network; proteins that may participate in these processes will be identified and their roles in vesicle budding will be tested in an in vitro assay that reconstitutes transport; (2) to study the components involved in vesicle targeting and fusion. GTP binding proteins and their accessory proteins involved in these processes will be identified and studied. Whether these factors are unique to the individual pathway will be determined; (3) to study the mechanism for sorting of proteins between the constitutive and the regulated pathways. Structural determinants important for sorting will be defined by mutagenesis studies. The current model for sorting will be examined by an in vivo test to determine if sorting of hormones into the regulated secretory pathway is achieved by formation of molecular aggregates.